Haemophagocytic lymphohistiocytosis due to Burkholderia pseudomallei in a primigravida

Introduction. Melioidosis is caused by Burkholderia pseudomallei, a Gram-negative, saprophytic bacillus, commonly found in soil or contaminated water. As infection with this bacterium produces a wide variety of clinical manifestations the organism is aptly called the ‘great mimicker’. Even though it is non-fastidious and an easily cultivable organism, it can be misidentified in automated identification systems. Case report. A 24-year-old primigravida presented with complaints of fever and myalgia of 45 days’ duration. She was diagnosed to have haemophagocytic lymphohistiocytosis (HLH) based on clinical and laboratory parameters. Blood and bone marrow culture sent to the microbiology laboratory grew non-fermenting Gram-negative bacilli which were misidentified as Burkholderia cepacia by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) technology. It was subsequently identified as B. pseudomallei by 16S rRNA gene sequencing. The patient was commenced on intensive phase therapy with intravenous ceftazidime for 2 weeks, followed by maintenance therapy with oral trimethoprim and sulfamethoxazole for 3 months. In view of HLH, she was treated with intravenous dexamethasone for 2 weeks which was later switched to oral dexamethasone for a period of 6 weeks. She responded well to the treatment, but had to undergo medical termination of her pregnancy as there was severe intrauterine growth restriction of the fetus. Conclusion. Prognosis of melioidosis is excellent if early diagnosis and appropriate antibiotic treatment is provided. In this era of automation, it is important to determine if the suspected pathogen is listed in the database of the automated identification system.


INTRODUCTION
Haemophagocytic lymphohistiocytosis (HLH) is a severe systemic inflammatory syndrome that can result in deregulated engulfment of haematopoietic cells by lymphocytes and activated macrophages.HLH may be inherited in an autosomal recessive manner or it can be acquired following various infections, malignancy, metabolic or rheumatological conditions, and is otherwise called secondary HLH.The development of secondary HLH in infections may be due to an inability to suppress or clear intracellular infections resulting in persistent activation of antigen presenting cells and CD8(+) T cells and increased production of cytokines.Secondary HLH is a potentially fatal complication of Burkholderia cepacia infection and has been reported in patients with chronic granulomatous disease [1,2].There are very few reported cases of HLH due to Burkholderia pseudomallei [3].As B. pseudomallei is a non-fastidious bacterium, it can be easily isolated from clinical samples, but can be misidentified in automated systems.We report a case of HLH due to B. pseudomallei, a facultative intracellular pathogen.

CASE REPORT
A 24-year-old primigravida was referred to the medicine department of the hospital with complaints of intermittent high-grade fever associated with chills and generalized body pain for the past 45 days.There was no other significant medical history.The patient was a home maker living in a coastal city in southern India.She had been evaluated as an inpatient for the current illness in two other healthcare facilities, with no conclusive diagnosis, before the present hospital admission.
Physical examination revealed the patient to be febrile with appreciable pallor.Per abdomen uterus size corresponded to 22 weeks of gestation.Examinations of other systems were unremarkable.Laboratory investigations revealed anaemia (haemoglobin: 7.3 g dl −1 , total white blood cell count: 9290 cells mm −3 , platelet count: 3.17×10 5 mm −3 ).Biochemical markers demonstrated a raised triglyceride level (3.1751 mmol l −1 ) and serum ferritin levels (1619 µg l −1 ) and normal liver function.Bone marrow biopsy showed reduced normoblastic maturation of all lineages, presence of haemophagocytes, normal granulopoiesis and normal megakaryocytes with absent iron stores.
Screening tests for autoimmune markers (rheumatoid arthritis factor, antinuclear/cytoplasmic antibodies) were negative.2D echo showed no signs of infective endocarditis.Magnetic resonance imaging (MRI) of the thorax was normal study.MRI of the abdomen revealed hepatosplenomegaly and a single live intrauterine gestation.The patient was diagnosed to have HLH, as she had five of the eight criteria needed for diagnosis, which included presence of fever, splenomegaly, haemophagocytes in bone marrow, raised serum ferritin and raised triglyceride levels.Detection of soluble IL-2 receptor levels and natural killer cell activity could not be done in the patient as these tests are not performed routinely in the laboratory.
Blood culture showed growth of non-fermenting Gram-negative bacilli which were presumptively identified as Acinetobacter species according to conventional biochemical tests but were found to be resistant to polymyxin B discs (300 units).
Blood and bone marrow samples were sent again for bacterial culture after 1 week.Colonies of non-fermenting Gram-negative bacilli grew on 5 % sheep blood agar (Fig. 1) and MacConkey agar (Fig. 2).Even though the bacteria did not ferment lactose, the colonies on MacConkey agar appeared pink after a few days due to uptake of the dye from the medium.It was found to be delayed oxidase-positive and was identified as B. cepacia with 30 % probability by matrix-assisted laser desorption ionization-time of flight MS (MALDI-TOF MS) (VITEK MS; bioMérieux).We further found that B. pseudomallei was not present in the database of the VITEK MS system.Further identification to the species level was done by PCR-based DNA sequencing of the 16S rRNA gene (GenBank accession number OP764052) and growth of B. pseudomallei was reported.It was found to be susceptible to ceftazidime, imipenem, meropenem, trimethoprim/sulfamethoxazole, amoxicillin/clavulanic acid and tetracycline.Antimicrobial susceptibility testing was done using the automated Vitek 2 COMPACT system (bioMérieux) with AST-N406 panel for Gramnegative bacilli, and the break points (MICs) were interpreted according to M45 CLSI (Clinical Laboratory Standards Institute)  The patient was initially started on intravenous piperacillin tazobactam, as per the blood culture susceptibility report of Acinetobacter species, which was then changed to intravenous ceftazidime 1 g 6-hourly for 2 weeks.In view of HLH, she was given intravenous dexamethasone 8 mg twice daily for 2 weeks, followed by oral dexamethasone 4 mg twice daily which was tapered and stopped over a period of 6 weeks.She received three units of packed cell transfusion as well as parenteral supplementation of iron for correction of anaemia.
The patient was counselled for termination of her pregnancy as there was severe intrauterine growth restriction (IUGR) of the fetus.Following stabilization of the patient's condition, medical termination of the pregnancy was done.The patient was symptomatically better and her fever resolved within 72 h of initiation of therapy .Considerable improvements in the clinical and laboratory parameters were observed in the first week of treatment.Hence, the intensive therapy was followed by maintenance therapy of oral trimethoprim/sulfamethoxazole 240 mg/1200 mg twice a day over a period of 3 months.Repeat haemoglobin testing 2 weeks later showed a raised haemoglobin level of 11.9 g dl −1 .

DISCUSSION
Melioidosis is endemic in northern Australia and Southeast Asian countries, especially Thailand and Malaysia, and is being increasingly recognized in other regions.The global incidence of human melioidosis is approximately 165 000 cases per year worldwide and South Asia has the highest burden of the disease (44 % of all cases).As the largest country in South Asia with a large diabetic population and favourable environmental conditions, India is a potential hotspot for the disease [4].
The patient in the present case report did not have any history of chronic granulomatous disease or cystic fibrosis nor did she undergo any invasive procedure or prolonged hospitalization for acquisition of B. cepacia infection.In this scenario, we sent the bacterial isolate for further identification by 16S rRNA gene sequencing.This case highlights that B. pseudomallei could be another possible causative bacterial agent of HLH.
Individuals with diabetes mellitus, renal disease or thalassaemia, or those undergoing immunosuppressive therapy and those with alcohol misuse are at increased risk of acquiring melioidosis.A review of local data in Malaysia showed that B. pseudomallei infection can occur in 15-42 % of individuals even in the absence of these risk factors [9].The patient mentioned in this case report was a young, immunocompetent primigravida.There is no clear evidence on antenatal outcomes with melioidosis, and it is not known if pregnancy is a risk factor for B. pseudomallei infection or if such individuals experience more severe disease [10].
A definitive diagnosis of melioidosis requires a positive culture of B. pseudomallei.The organism can be misidentified as Pseudomonas spp. or Acinetobacter spp.if a detailed biochemical analysis is not performed.Commercially available identification systems and automated identification algorithms may misidentify B. pseudomallei as other Burkholderia spp.such as B. cepacia complex, Chromobacterium violaceum and Pseudomonas spp.However, the pattern of resistance to antimicrobials is distinctive as most B. pseudomallei isolates are resistant to aminoglycosides and polymyxin B .
Molecular methods such as 16S rRNA gene sequencing are widely used for identification of bacteria to the species level in clinical microbiology laboratories and are applicable to B. pseudomallei also.Other gene targets used for identification of B. pseudomallei include groEL and the type III secretion system [11].
Detection of specific antibodies can be done in cases of chronic melioidosis, but acutely bacteraemic patients appear less likely to have antibodies [11].Antigen detection tests can be done directly on clinical specimens (e.g.sputum, urine or pus), but the sensitivity is significantly lower than with culture [12].
Treatment for melioidosis includes two phases, an initial intensive phase and subsequent eradication phase.The intensive phase includes the use of intravenous ceftazidime and carbapenem (either meropenem or imipenem) for at least 10-14 days.The addition of trimethoprim sulfamethoxazole for a longer duration of the intensive phase is required for those patients who are critically ill or in patients with extensive pulmonary disease, organ abscesses, osteomyelitis, septic arthritis and neurological melioidosis.The eradication/maintenance phase is for a minimum of 3 months and the preferred drug is trimethoprim sulfamethoxazole [13].Alternative drugs used in eradication therapy include amoxicillin-clavulanic acid, oral quinolones (ciprofloxacin, levofloxacin), doxycycline and chloramphenicol.Amoxicillin-clavulanic acid is preferred in pregnant women and in those who are intolerant of trimethoprim sulfamethoxazole.Standard therapy for HLH involves administration of steroids, cyclosporin and etoposide.Steroids are added to the antimicrobial agent when the bacterial cause for HLH has been identified.

CONCLUSION
B. pseudomallei can be a rare cause of HLH.Early and accurate identification of the organism is essential for optimal treatment as it requires prolonged antibiotic therapy that includes an intensive phase and maintenance phase.Its important to ensure the suspected organism is present in the database of the automated identification system.Collaboration between the treating physician and the microbiologist is vital in improving the outcome in patients with HLH.
5. This organism is found near coastal areas; a line may be added about the residence of the patient.Though at present there are reports of B. pseudomalliei infection from the central area of India also.
Added the place of residence 6.Lines 28-29: The authors stated that it was misidentified as B. cepacia.Looking at the typical colony morphology, a reliable guess can be made about the differential diagnosis of dry colonies producing organisms and B. cepacia isn't amongst those two/ three organisms.
It was misidentified in the automated MALDI TOF system.Through the case report we wanted to highlight that in few cases the automated identification systems may misidentify B.pseudomallei.Gram negative bacilli (Typical safety pin appearance could not be appreciated.)Triple disc screening was not done for the isolate.
3.Was the duration of intensive phase (16days) sufficient, keeping in mind the clinical presentation and concurrent steroid therapy The patient had resolution of fever in 72 hours of initiation of IV ceftazidime and considerable improvement in all parameters in first 2 weeks, hence stepped down to maintenance therapy.

4.Any follow up laboratory findings of CBC, prior to starting Co-trimoxazole, which also carries a risk of cytopenia
The patient did not have cytopenia prior to starting Co -trimoxazole (Total WBC count:8840/mm 3 ,RBC count 2.75 million/ mm 3 ,platelet count : 3.17 lakhs/ mm 3 and repeat counts after starting therapy were also within normal limits.

incomplete Literature review and failure to cite prior reports of Hemophagocytic lymphohistiocytosis due to Burkholderia pseudomallei
We could come across only 1 case which has been enclosed.Comments: Thank you for submitting your manuscript for publication in Access Microbiology.It has been examined by expert reviewers who have concluded that the work is of potential interest to the readership of Access Microbiology.However, based on the comments received, it is clear that a major revision of this manuscript will be required before a decision can be made on its publication.I will be pleased to consider a revised manuscript along with a document including a point by point response to each of the reviewers comments.Your revised manuscript may be returned to one or more of the original reviewers, along with your itemised response to the reviewers' comments.I look forward to receiving the revised manuscript.

Reviewer 2 recommendation and comments
https://doi.org/10.1099/acmi.0.000520.v1.3 © 2022 Anonymous.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.

Date report received: 24 July 2023 Recommendation: Major Revision
Comments: The case report Hemophagocytic lymphohistiocytosis due to Burkholderia pseudomallei in a primigravida was reviewed.Though an interesting observation, several points need to be addressed 1.How many points of the (Henter et al Criteria 2004) were fulfilled for arriving at a diagnosis, Could it be possible Hemophagocytic lymphohistiocytosis 2. What were the results of bench side tests, Gram stain, triple disc screening 3. Was the duration of intensive phase (16days) sufficient, keeping in mind the clinical presentation and concurrent steroid therapy 4. Any follow up laboratory findings of CBC, prior to starting Co-trimoxazole, which also caries a risk of cytopenia 5. incomplete Literature review and failure to cite prior reports of Hemophagocytic lymphohistiocytosis due to Burkholderia pseudomallei

Please rate the quality of the presentation and structure of the manuscript Satisfactory
To what extent are the conclusions supported by the data?Partially support

Is there a potential financial or other conflict of interest between yourself and the author(s)? No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?Yes Reviewer 1 recommendation and comments https://doi.org/10.1099/acmi.0.000520.v1.4

Guidelines 2018 .
Currently there are disc diffusion guidelines in EUCAST for interpreting susceptibility of B. pseudomallei to antimicrobial agents.

7 . 2 1.
Lines 130: Which antibiotic was specifically to this patient and for how long?Please describe it discreetly.Ceftazidime (intravenous): 1 gm 6 th hourly for 2 weeks Trimethoprim/sulfamethoxazole (oral 240/1200) twice a day for 3 months 8.It needs linguistic improvement and the minor grammatical errors to be corrected.Revised Reviewer How many points of the (Henter et al Criteria 2004) were fulfilled for arriving at a diagnosis, Could it be possible Hemophagocytic lymphohistiocytosis Five of the eight criteria has been fulfilled, which is required for the diagnosis for HLH (Revised Henter criteria 2004) Detection of soluble interleukin-2 receptor levels and natural killer cell activity could not be done in our laboratory 2. What were the results of bench side tests, Gram stain, triple disc screening

1
Editor recommendation and commentshttps://doi.org/10.1099/acmi.0.000520.v1.5 © 2022 Allen D. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.Danielle Allen; Queen's University Belfast, School of Biological Sciences, UNITED KINGDOM, Belfast Date report received: 24 July 2023 Recommendation: Major Revision